Nitric oxide is a paramagnetic molecule. The detection of NO in biological tissues and liquids is a challenge because of its low concentration and small half-life.
A proper assay of the level of bioavailable nitric oxide in vivo in human circulation is important for the protection of human health. Insufficiency of NO production from the endothelium is a crucial sign of endothelial dysfunction in many metabolic diseases and, especially, cardiovascular diseases developed under various risk factors including age, hypertension, smoking, and hypercholesterolemia. The functionality of the endothelial nitric oxide synthase (eNOS) and NO bioavailability in the vascular bed in vivo are difficult to assess quantitatively, especially in humans.
Invasive and non-invasive methods were developed to indirectly determine the level of bioavailable NO and study its dynamics. The level of eNOS expression, the phosphorylation status of eNOS and eNOS activity can be studied ex vivo in biopsy samples from isolated vessels and tissues. However, this analysis cannot characterize NO bioavailability in vivo.
NO can be converted into nitrite and nitrate in reactions with various specific and non-specific targets. Therefore, measurements of nitrite/nitrate (NOx) concentration in extracellular fluids such as blood plasma have been widely used in different laboratories. The Griess reaction detects nitrite based on its reaction with sulphanilic acid. The reaction product is then detected by spectrophotometry. The chemiluminescence technique provides higher sensitivity using back-reduction of nitrite/nitrate to NO in a reflux chamber at 95° C. Both techniques allow nitrite/nitrate detection in biological samples, however, proper data interpretation is difficult due to the dietary variability and the active nitrite/nitrate metabolism. Moreover, peroxynitrite, a strong oxidant produced by the reaction of superoxide anions and NO and detected at high level in pathophysiological models, is also converted to NOx as end product.
The level of NO production can be detected amperometrically by NO-specific electrodes. The method requires catheterization in order to insert the electrode close to the endothelium, which is very fragile and can be damaged by this invasive procedure and the application of the method is limited by the sensitivity/specificity of the signal depending on the sensor.
EndoPAT is a standardized device for non-invasive endothelial function assessment. The technology is based on the detection of peripheral arterial tone signal using volume-sensitive sensors placed on the fingertips. As the endothelial vasodilation is mediated by several other factors besides NO, the EndoPAT method can only be used as an indirect assessment of endothelial NO production.
Nitric oxide is known to bind tightly to hemoglobin (Hb). Interactions of NO with Hb are believed to be a major route of NO metabolism in the vascular bed. It follows that the levels of Hb-NO in blood are an excellent indication of endogenous NO production.
Distinct forms of paramagnetic heme-NO adducts can be formed depending on NO hosting at α- or β-subunits and the Hb conformation state under variation of oxygen pressure and allosteric factor abundance in erythrocytes. Actually, three forms of nitrosylated Hb with the same principal g-values (gxx=2.08, gyy=2.04, and gzz=2.01) can be observed in erythrocytes in human blood: i) 5-coordinated nitrosylated α-Hb (T-form, deoxy-like); ii) 6-coordinated nitrosylated α-Hb (R-form, oxy-like); iii) 6-coordinated nitrosylated β-Hb (R-form, oxy-like). Remarkably, the EPR spectrum of T-form (deoxy-like) displays the well-resolved hyperfine structure (Azhfs=16.8 G) due to the net donation of electron density from Fe(II) to NO after cleavage of the bond between iron and proximal His of the R form. Moreover, the dissociation rate constant was also found to be much higher for the T-form than for the R-form (with a resulting t1/2˜15 minutes and 20 hours, respectively). This highlights the interest to follow the evolution of the T-form in venous blood as a dynamic marker of NO availability in the systemic circulation.
Electron Paramagnetic Resonance (EPR) is a method for the individual detection of the level of various paramagnetic compounds in biological samples. EPR is an extremely attractive technique to analyze NO bioavailability. Measurement of paramagnetic hemoglobin-nitric oxide adducts (Hb-NO) in whole blood and erythrocytes by the EPR spectroscopy was proposed in the last decade in animal models and human blood. Unique information about systemic NO levels could be obtained especially in animal models.
However, the EPR spectra of whole venous human blood showed a number of additional paramagnetic species with wide-line width assigned to Cu-containing proteins, such as ceruloplasmin, and narrow-line width signal with g˜2 assigned to protein-centered free radicals formed in the erythrocytes. The superposition of the EPR signals from different paramagnetic centers in the same region (g˜2) where the Hb-NO signal is observed made the Hb-NO quantitation problematic. The background signal of ceruloplasmin could be eliminated by separating the plasma from the erythrocytes while the free radical signal still represents a background signal that interferes with Hb-NO quantitation in vivo in human blood.
Hence, there remains a need in the art to eliminate the free radicals background in order to have an accurate measurement of NO using EPR spectroscopy. The present invention aims to provide a solution to at least the above-mentioned problem by providing a method for an accurate NO quantification in vivo in human blood.